The delivery of proteins to the cytosol has been difficult to achieve due to the barrier of the plasma membrane or the inefficiency of endosomal release, while many bacterial toxins have evolved the machineries to transport their catalytic components to the cytosol of mammalian cells. Anthrax toxin is one example that uses three components1: the translocase protective antigen (PA) and two enzymatic factors, Lethal Factor (LF) and Edema Factor (EF)2. PA binds to receptors on host cells3,4 and is cleaved by a furin-family protease5,6. The resulting fragment PA63 self-assembles to the ring-shaped heptameric and octameric prepore7,8, forming complexes with LF and EF with high affinity. The complexes are then endocytosed to the endosome, where the acidification triggers the conformational rearrangement of prepore to form an ion-conductive β-barrel transmembrane pore. The pore then translocates LF and EF to the cytosol to act on their selective target proteins9,10 (FIG. 6). FIG. 6 is a model of anthrax toxin entry into cells.
LF binds to the surface of PA through the 263-residue N-terminal domain (LFN) with nanomolar affinity. The crystal structure of PA8 (LFN)4 prepore complex shows that the first α-helix and β-strand (α1β1) of LFN unfold and dock into the cleft between two adjacent PA subunits, called a clamp, providing the translocase a key handle to grip the substrate.11 LFN is further partially unfolded under the acidic pH in the endo some and its N terminus binds to the hydrophobic heptad of F427 residues in PA63 pore, called φ-clamp, initiating the unfolding and translocation of the protein in an N- to C-terminal direction through the narrow β-barrel channel.12-16 
Both the α clamp and φ-clamp interact with a broad array of amino acid sequences, providing the wide chemical complexity and configurational flexibility to the PA63 pore. LFN fusions of the A chain of diphtheria toxin (DTA), catalytic domains of pseudomonas exotoxin A and Shiga toxin,17,18 and some other proteins have been recombinantly expressed and successfully translocated through PA63 pores. A more recent study has shown the effects of incorporation of D-amino acids and cysteic acid at the N-terminus of LFN on translocation through the pore.19 The ability of the PA63 pore in translocating non-natural chemical entities has not been well exploited.